Sealant or tissue generating product

ABSTRACT

The present invention is related to a sealant or a tisuue generating product comprising a (coagulated) plasma matrix, one or more growth factors, at least one phospholipid and a protein scatffold for the generation of said tissue (or the coagulation factor VII).

FIELD OF THE INVENTION

The present invention relates to a sealant or a tissue (a hard tissuesuch as bone or cartilage, or a soft tissue, such as skin or theepithelial tissue of the stomach) generating product comprising a(coagulated) plasma matrix, one or more growth factor(s), at least onephospholipid and a protein scatffold for the generation of said tissue(or the coagulation factor VII).

BACKGROUND OF THE INVENTION AND STATE OF THE ART

Many researches have been made for the preparation of tissues substituteor implant.

For instance, for the preparation of bone substitute or implant, it isknown to treat human bone by chemicals for destroying prions. The sotreated human bone acts as a porous matrix suitable for the growth ofcells after its implant.

It has also been proposed to prepare artificial matrix or sponge fromcollagen containing material and to use said matrix or sponge as bonesubstitute.

Example 4 of U.S. Pat. No. 5,733,545 discloses the preparation of clotfrom a mixture containing a plasma-buffy coat concentrate and ground drybone or from a plasma-buffy coat concentrate and CaCl₂, said lattercompound being used for ensuring the coagulation of the mixture. In saidexample 4, it is stated that the chelation of the plasma-buffy coatconcentrate containing ground dry bone is possibly due to the presenceof calcium from the solid bone. In said example, it is clearlystipulated that the use of thrombin is a cause of patient complications.

However, the bone substitute obtained by mixing a plasma-buffy coatconcentrate and ground dry bone was not suitable for the bonegeneration.

For inducing bone repair, Friadent is commercializing the product“PEPGEN 15” and “pepgen 15 Flow” which consist of a synthetic resorbablematrix containing a synthetic amino acid peptide of formulaGTPGPQGIAGQRGVV (PEPGEN15), and inorganic particles containing calciumphosphate and having a mean particle size lower than 750 μm.

The following table gives the correlation between the amino acid, itsone-letter code and its three letter code. One-letter Three-letter AminoAcid Symbol Symbol Alanine A Ala Arginine R Arg Asparagine N AsnAspartic acid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E GlnGlycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine KLys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S SerThreonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

This product enables good histologic regeneration in 2/4 casesevaluated.

SUMMARY OF THE INVENTION

The present invention is related to a tissue generating productcomprising a (coagulated) plasma matrix, (preferably a (coagulated)matrix of platelet rich plasma, or a (coagulated) matrix of plateletpoor plasma), one or more growth factor(s)), preferably a recombinantcompound for generating thrombin, at least one phospholipid and aprotein scatffold for the generation of said tissue.

The protein scatffold present in the tissue generating product accordingto the invention is chosen according to the type of tissue which shouldbe regenerated. This tissue could be a hard tissue (such as a bone orcartilage) or a soft tissue (such as skin or an epithelial tissue of thestomach). Preferably, the tissue generating product according to theinvention is a bone generating product which comprises as a proteinscatfold for the generation of said tissue a bio-engineerated osteoinductive bone substitute, such as the compound PEPGEN P15™ or PEPGENP15 Flow (sold by the Company CERAMED) and which comprises an inorganicbovine bone material (as said protein scatfold for the generation ofsaid bone tissue) and a peptide of 15 amino acid obtained from collagentype I involved in adhesion of reparative cell and having the formulaGTPGPQGIAGQRGVV.

Another example of such bio-engineerated osteo inductive bone substituteis the compound OSIGRAFT™, (sold by the Company STRIKER) which is a pureresorbable protein scatffold obtained from bovine collagen comprisingHeptotermin a (recombinant osteogenic protein 1), which is a bonemorphogenetic protein 7 produced in CHO cells and which initiates boneformation through induction of cellular differentiation in mesenchymalcells.

Another example of such bio-engineerated osteo inductive bone substituteis the compound Ossigel™ (sold by the Company ORQUEST) which comprisesthe compound Hyaluronic acid which is a viscoelastic polymer complementto the activity of the tissue factor bFGF (basis fibroblast growthfactor) which is advantageously a mytogen and potent angiogenic factor.

A further example of such bio-engineerated osteo inductive bonesubstitute is the compound Infuse™ (sold by the Company MEDTRONIC) whichis a collagen sponge and which comprises a recombinant human bonemorphogenetic 2 (rhBMP2) which could be used for the stimulation of newbone formation.

A last example of said protein scatffold is a matrix of collagen,reticulin and/or elastin fibers or their precursors (tropocollagen,tropoelastin, . . . ).

The protein scatffold for the bone tissue generation used in the tissuegenerating product according to the invention is an artificial matrix orsponge which further comprises one or more tissue factors, as well asother elements that facilitate the tissue regeneration, such aseffective amount of calcium containing compound used for regeneration ofa bone tissue.

Advantageously, the tissue generating product according to the inventioncomprises a matrix of coagulated platelet rich plasma with a highconcentration of platelets (GERNOT WEIBRICH et al, Clin. Otal. Impl.Res. 13, p. 437-443, 2002; GERNOT WEIBRICH et al, Clin. Otal. Impl. Res.14, p. 357-362, 2003). The platelet concentration (comprising differentgrowth factors) will advantageously present an improved influence uponthe regeneration of the tissue.

Advantageously, a coagulated matrix of platelet rich plasma has aplatelet concentration higher than 1,500,000 platelets per microlitre ofthe matrix forming agents.

Preferably, said platelet rich plasma has a platelet concentrationcomprised between 1,500,000 platelets and 20,000,000 platelets permicrolitre of the matrix forming agents.

Preferably, said concentration is comprised between 2,000,000 plateletsper microlitre and 8,000,000 platelets per microlitre of the matrixforming agents, more preferably about 5,000,000 platelets permicrolitre.

The tissue generating product may also comprise a coagulated matrix ofplatelet poor plasma, which means that the platelet concentration isadvantageously lower than 1,500,000 platelets per microlitre of thematrix forming agents, preferably the platelet concentration is lower500,000, 100,000 or 50,000 platelets per microlitre of the matrixforming agents.

For the preparation of the tissue generating product it is possible toobtain from a single blood sample, preferably obtained from the bloodpatient an autologous platelet rich plasma matrix suitable for a firstapplication and from the remaining material sample, an autologousplatelet poor plasma matrix suitable for a second application of thetissue generating product according to the invention.

According to the type of application, the tissue product could alsocomprise other elements, such as an effective amount of calciumcontaining compound dispersed in said matrix (for inducing the formationof a hard tissue, such as bone).

Said calcium phosphate containing compound is selecting from the groupconsisting of synthetic hydroxyapatite, CaCl₂, β-tricalcium phosphate,bone particles (denatured bone), bone particles (not denatured bone),apatite, aspidine, calcium sulfate, calcium carbonate, hydroxyapatite,(from coral reef), calcium gluconolactate, calcium gluconate, calciumlactate, calcium glutonate and mixtures thereof. (Preferably, saidcompound having no sharp or pointed edges).

Said effective amount of calcium containing compound could be inorganicparticles containing calcium phosphate and having a mean particle sizelower than 750 μm.

In the tissue bone generating product, the inorganic particlescontaining calcium phosphate have preferably a mean size comprisedbetween 150 μm and 500 μm.

The bone particles comprised in the bone generating product according tothe invention are preferably selected from the group consisting ofcraniofacial bone particles, iliac bone particles and mixture thereof.Said bone particles are preferably derivated from non denatured bonesand the calcium phosphate containing particles have substantially nosharp and no pointed edge.

Advantageously, the bone particles have an average particle sizecomprised between 0.5 mm and 5 mm, preferably comprised between 0.5 and3mm, most preferably about 1 mm (average in weight). The bone particleshave for example the form of chips or flakes having an average particlesize comprised between 0.5 mm and 5 mm, preferably comprised between 0.5and 3 mm, most preferably about 1 mm (average in weight). According to apossible embodiment, the bone particles consist of a mixture ofdenatured bone particles (for example bone particles prepared bygrinding a bone that has been treated by chemical(s), by irradiation,etc. for rendering it prion free.) and of not denatured bone particles.When using some denatured bone particles, the said particles ofdenatured bone can have a particle size lower than 0.5 mm, as saiddenatured bone particles are used to add some calcium to the product.

The tissue generating product of the invention comprises for examplefrom 5% to 50% by volume of bone particles, advantageously from 10 to40%, preferably from 20 to 30% by volume of bone particles. The boneparticles forms preferably more than 90% by weight of the calciumcontaining compound present in the tissue generating product of theinvention.

Furthermore, the tissue generating product according to the inventionmay also comprise further elements such as buffer agents, antibiotics,additives (selecting from the group consisting of growth factors, genesencoding growth factors, drugs, fatty acids, bactericides or virucides)and compounds for inducing the formation of matrixes and mixturesthereof.

Preferably, the said antibiotics have an anti-osteoclast effect.

Said additional element are preferably growth factor(PDGFAA,PDGFAB,PDGFBB, superfamily BTGF and family of BMP, such asBMP-1, etc.), gene coding BMP and/or BTGF, steric factors, calciumcontaining compounds, drugs, fatty acids, antibiotics or mixtures ofantibiotics (preferably compound(s) having an anti-osteoclast effect,such as antibiotics of the tetracyclin group, Vibramycin®, Doxycycline®,Minocycline, Minocin® (Wyeth-Lederlee), and mixtures of compound(s)having an anti-osteoclast effect with another antibiotic(s), such asmacrolide, penicillin based compounds, etc.), bactericide, virucide,fibrinogen, compounds inducing the formation of a matrix, buffer,zwitterionic buffer system at physiological pH, etc. and mixtures ofsaid compounds or additives.

According to a detail of a preferred embodiment, the tissue generatingproduct contains from 0.001 to 10% by weight antibiotic or antibiotics(calculated in its dry form), advantageously from 0.01% to 5% by weight,preferably from 0.02 to 1%, for example from 0.05 to 0.4% by weight. Theantibiotic is advantageously selected from the group consisting ofantibiotics having an anti-osteoclast effect (more specificallyantibiotics of the tetracyclin group, Vibramycin®, Doxycycline®,Minocycline, Minocin® (Wyeth-Lederlee)), mixtures of antibiotics havingan anti-osteoclast effect, and mixtures of one or more antibioticshaving an anti-osteoclast effect with one or more other antibiotic(s)(preferably macrolide, penicillin based compounds, etc. and mixturesthereof).

Before its gelling, the tissue generating product has advantageously apH substantially equal to the physiological pH, for example a pHcomprised between 6.5 and 8, preferably about 7-7.5, pH measured at 37°C.

Among the recombinant growth factors present in the compositionaccording to the invention, one may select (recombinant, which meansthat said growth factors do not present contaminants) tissue factorshaving no membrane binding sequence or having only an extra-cellulardomain.

Preferably, said growth factors are selected from the group consistingof the human (recombinant) tissue factor (rhTF), the human (recombinant)platelet-derived growth factor (rhPDGF), the human (recombinant)transforming growth factor (rhTGF), the human (recombinant) insulin-likegrowth factor (rhIGF), the human (recombinant) epidermal growth factor(rhEGF), the human (recombinant) hepatocyte growth factor (rhHGF), . . ..

According to a preferred embodiment, the recombinant compound forgenerating thrombin (or tissue factor) is combined, preferably mixed,with at least a phospholipid selected from the group consisting ofphosphatidylserine, derivatives thereof, phosphatidylserine having atleast one fatty acid side chain, phosphatidylcholine, derivativesthereof, phosphatidylcholine having at least one fatty acid side chain,and mixtures thereof. Preferably, the recombinant compound forgenerating thrombin (or the tissue factor) is combined with at least aphospholipid selected from the group consisting of phosphatidylserine,derivatives thereof, phosphatidylserine having at least one fatty acidside chain, phosphatidylcholine, derivatives thereof,phosphatidylcholine having at least one fatty acid side chain, andmixtures thereof, the fatty acid side chain being selected from thegroup consisting of fatty acid chains with at least one double bond andwith 6 to 24 carbon atoms, most preferably with 16 to 18 carbon atoms.

The tissue generating product according to the invention could also be asealant which comprises said mentioned plasma matrix, one or more ofsaid mentioned recombinant factor(s), (preferably a recombinant compoundfor generating thrombin) in presence of at least one of said mentionedphospholipid, preferably two different phospholipids and as a proteinscatffold being the coagulation factor VII (FVII) which improves thesealant characteristics.

A further aspect of the present invention is related to a kit for thepreparation of a sealant or a tissue generating product prepared bycontacting said plasma matrix with a recombinant compound for generatingthrombin (or growth factor), in presence of at least one phospholipid(preferably at least two different phospholipids), a protein scatffoldfor regeneration of said tissue or the coagulation factor VII andpossibly an effective amount of calcium containing compound dispersed insaid matrix for inducing the formation of the tissue being a bone(preferably said compound being inorganic particle containing a calciumphosphate having a mean particle size lower than 750 μm). Said kitcomprising at least one system selecting from the group consisting of avial containing, a recombinant compound for generating thrombin (orgrowth factor), the protein scatffold element or coagulation factor VII,and possibly an effective amount of calcium containing compounds forinducing the formation of the tissue being a bone (preferably inorganicparticles containing calcium phosphate and having a mean particle sizelower than 750 μm); two distinct vials, a first containing a recombinantcompound for generating thrombin (or growth factor) while the secondvial contains the protein scatffold element or the coagulation factorVII and possibly said effective amount of calcium containing compoundsfor inducing the formation of a tissue being a bone (preferablyinorganic particles containing calcium phosphate and having a meanparticle of size lower than 750 μm). The vial may also comprise theother elements present in the product according to the invention, suchas a vial containing at least one buffer agent and at least oneantibiotic and one or more of the other additives above mentioned.

The antibiotic formulation could be an oral antibiotic formulation,injectable antibiotic formulation, topic antibiotic formulation, sprayantibiotic formulation and inhalated antibiotic formulation, saidformulation being suitable for administrating to the patient anefficient dose of antibiotics at the place where the tissue has to beregenerated. Oral formulation and injectable formulation are preferred.

According to a detail of a preferred embodiment, the amount ofantibiotic or antibiotics added to the sealant or tissue generatingproduct or used during the coagulation of the matrix plasma tissuegenerating product or administered prior, during and/or after theapplication of the sealant or tissue generating product to the patientis such that the tissue generating product contains from 0.001 to 10% byweight antibiotic or antibiotics (calculated in its dry forms),advantageously from 0.01% to 5% by weight, preferably from 0.02 to 1%,for example from 0.05% to 0.4% by weight, more specifically from 0.2 to0.3%. The antibiotic is advantageously selected from the groupconsisting of antibiotics having an anti-osteoclast effect (morespecifically antibiotics of the tetracyclin group, Vibramycin®,Doxycycline®, Minocycline, Minocin® (Wyeth-Lederlee)), mixtures ofantibiotics having an anti-osteoclast effect, and mixtures of one ormore antibiotics having an anti-osteoclast effect with one or more otherantibiotic(s) (preferably macrolide, penicillin based compounds, etc.and mixtures thereof).

Most preferably, at least one antibiotic is added to the mixturecontaining at least platelet rich plasma and calcium phosphatecontaining compound, before adding the recombinant compound thatgenerates thrombin, but also advantageously when adding the recombinantcompound that generates thrombin.

When no antibiotic is added to the mixture or when a low amount ofantibiotic is added to the mixture, antibiotic(s) are advantageouslygiven to the patient by oral administration, by injection, by topicapplication, by inhalation, preferably by oral administration and/or byinjection (most preferably injection in the blood or percutaneousinjection), prior and/or during and/or after the application of the bonegenerating product to the patient.

Preferably, the patient is submitted to at least one treatment with atleast one antibiotic, said treatment being selected from the groupconsisting of:

-   -   oral administration of an efficient dose or effective amount of        at least one antibiotic at least after the application of the        sealant or tissue generating product to the patient;    -   oral administration of an efficient dose or effective amount of        at least one antibiotic at least prior the application of the        sealant or tissue generating product to the patient;    -   injection of an efficient dose or effective amount of at least        one antibiotic at least after the application of the sealant or        tissue generating product to the patient;    -   injection of an efficient dose of at least one antibiotic at        least prior the application of the sealant or tissue generating        product to the patient;    -   administration of an efficient dose or effective amount of at        least one antibiotic at least for one day prior the application        of the bone generating product and at least for one day after        the application of the sealant or tissue generating product to        the patient.

According to a specific embodiment, an effective amount of antibiotic isadministered (most preferably orally or by injection) to the patientbefore the application of the sealant or tissue generating product ofthe invention, as well as after said application.

The invention relates also to a method for the preparation of thesealant or tissue generating product according to the invention in which:

-   -   a substantially homogeneous mixture is formed by mixing the        plasma matrix (preferably platelet rich plasma or platelet poor        plasma) with an effective amount of the protein scatffold or        coagulation factor VII and possibly calcium containing compound        for inducing the tissue generation when adding to the mixture of        a growth factor such as a recombinant thrombin generating        compound and at least one phospholipid,    -   the tissue factor or a recombinant thrombin generating compound        and at least one phospholipid are added and mixed to the mixture        of protein scatffold and coagulation factor VII and possibly        calcium containing compound (such as bone particles or        hydroxyapatite) and plasma matrix (preferably platelet rich        plasma or platelet poor plasma), and    -   the said mixture is kept under conditions for ensuring a        coagulation of the plasma matrix and the formation of the        sealant or tissue generating product.

Preferably, the coagulation of the matrix is carried out in presence ofoxygen and substantially without stirring. The said coagulation is mostpreferably carried out at a temperature comprised between 35° C. and 40°C., more specifically at a temperature of about 37° C.

Advantageously, at least two different phospholipids are added to themixture, said addition being preferably carried out when adding therecombinant thrombin generating compound (or tissue factor).

In the process of the invention, the recombinant thrombin generatingcompound (or tissue factor) is advantageously combined with thephospholipid (above described), preferably with two phospholipids, thesaid compound combined with phospholipid(s) having advantageously theform of a lyophilized product, such as a lyophilized cake, powder orgranules.

According to a detail of a preferred method of the invention, thecoagulation of the plasma matrix is carried out in presence of at leastone antibiotic or at least one antibiotic is added to the mixture afterthe coagulation of the matrix. The antibiotic or mixture of antibioticscan possibly be added to the other elements (the bone particles and/orthe bone before its grinding and/or to the recombinant compound thatgenerates thrombin or tissue factor and/or to a phospholipid).

In a preferred method of the invention, a gel (most preferably ahydrogel) is advantageously formed by the contact (preferably themixing) of the plasma matrix with a recombinant compound for generatingthrombin (or tissue factor) in presence of at least one phospholipid andthe protein scatffold or the coagulation factor VII and possibly theinorganic particles, whereby during the gel formation, the pH of theplasma matrix is kept between 6 and 8.

The gel can also be formed in presence of at least one buffer agent.Possible buffers are for example TRIS buffer, solution of Ringe, sodiumbicarbonate, and mixture thereof.

A last aspect of the present invention is related to a method forgenerating a tissue (such as bone) in a mammal patient (including thehuman) in need, said method comprising the step of applying at the placewhere the tissue (bone) has to be generated the sealant and/or tissuegenerating product according to the invention.

A last aspect of the present invention is also related to the use of thesealant and/or the tissue generating product according to the inventionfor the manufacture of a medicament in the treatment of tissue damagesin a mammal patient (including the human).

The preferred characteristics of the various aspects of the presentinvention will be described in details in the enclosed examples.

DESCRIPTION OF EXAMPLES

For the preparation of the said examples, the following products havebeen used:

PRP: platelet rich plasma of the patient to which a bone graft has to beplaced. The platelet concentration of the plasma was 1,800,000 plateletsper microliter of the plasma (higher concentration of platelets can alsobe used). The PRP was subjected to known usual treatments for theremoval leucocyte, for obtaining a maximum proportion of livingplatelets, for bacteriological control, said PRP being active at leastfor 5 days. Prior its use, the PRP was shaken at a temperature of 37°C., the said shaking being achieved by shaking the container containingthe PRP.

Thromboplastin: the thromboplastin used was a thromboplastin sold underthe Trademark Innovin by the company DADE AG (Düdingen, Switzerland).The thromboplastin is a recombinant human tissue factor lyophilizedcombined with synthetic phospholipids, namely phosphatidylserine andphosphatidylcholine, said phospholipids having at least one fatty acidside chain, the fatty acid side chain being selected from the groupconsisting of fatty acid chains with at least one double bond and with16-18 carbon atoms. Innovin is free of prothrombin, free of factor FVII,and free of factor FX. Calcium is present in Innovin. Innovin is a knownproduct for diagnostic purposes. Innovin contains also some calcium, azwitterionic buffer system at physiological pH. Innovin thromboplastincomprises a mixture of tissue factor and phospholipids, with a weightratio tissue factor/phospholipids of about 1/300. (molar ratioTF/phospholipids of about 1/10,000).

Other thromboplastin can also be used, such as thromboplastin sold byAmerican Diagnostica or thromboplastin developed by Henogen S A. Thethromboplastin developed by Henogen S A comprises a tissue factorwithout membrane binding sequence and without intracellular domain. TheHenogen tissue factor comprises thus only extra cellular domain. TheHenogen tissue factor was expressed by yeast and recovered as solubleand glycosylated form in the culture medium. The tissue factor waspurified by chromatography (in one or two steps, for example in twosteps). The thromboplastin formulation of Henogen comprises alsophospholipids, phosphatidyl-serine and phosphatidylcholine with a weightratio phosphatidyl-serine/phosphatidylcholine of 3:7. The molar ratioTF/phospholipids is about 1/1000. The recombinant tissue factor is mixedwith said phospholipids.

Bone particles: the bone particles have been prepared from iliac bonesor craniofacial bone of the patient to whom a bone graft is needed. Thefresh bone of the patient was ground in bone flakes (bone meal) havingan average diameter of 1 mm. The bone particles are added to the PRPjust after their preparation.

Water: water used is distilled, sterilized, pyrogen free water.

PepGen P-15®: this product sold by Friadent, Germany is a peptideenhanced by natural hydroxyapatite. The peptide has the formulaGTPGPQGIAGQRGVV. Said peptide is bound to natural calcium phosphateparticles (hydroxyapatite) with a size comprised between 250 μm and 420μm. The particles are anorganic bone mineral heated at a temperaturehigher than 1100° C. The particles have been submitted to a rolling soas to remove sharp and pointed edges. The weight ratio peptide offormula GTPGPQGIAGQRGVV/calcium phosphate particle is about250/1,000,000 or 0.00025. The particles are at least partly coated withsaid peptide.

PepGen P-15 Flow®: This product is similar to the product disclosed hereabove, except that it contains a resorbable gel matrix (made of sodiumsalt of a polycarboxymethyl ether of cellulose, glycerol and hydrogel).

Example 1

In said example, 50 ml of PRP was placed in a sterilized container. Avolume of 50 ml of PepGen P-15 or another protein scatfold abovedescribed was added to the PRP and mixed. The recipient is then heatedunder sterile conditions at 37.5° C. (for example by using a water bathhaving a temperature of 37.5° C., the said bath containing water and0.9% NaCl), in an oxygen containing atmosphere.

10 mg Innovin was mixed with 2 ml distilled, sterile and pyrogen freewater. The mixture water+Innovin was added to the PRP+PepGen P-15mixture kept at a temperature of 37.5° C.

After about 10 minutes, without stirring, a gel is formed in therecipient, said gel being a bone generating product suitable for implantto the patient.

Example 2

Example 1 has been repeated, except that 20 mg Innovin was mixed with 2ml distilled, sterile and pyrogen free water, and was added to themixture PRP+PepGen P-15.

Examples 3 to 9

In said examples, example 1 was repeated except that the amount ofreagents used was different. Example 3 4 5 6 7 8 9 PRP (ml) 50 50 50 5050 50 50 PepGen 35 35 45 40 60 75 75 P-15 (ml) Innovin 20 10 5 10 10 2010 (mg) Water (ml) 2 2 2 2 2 4 4

Examples 10 to 19

In said examples, example 1 was repeated except that the amount ofreagents used was different. Example 10 11 12 13 14 15 16 17 18 19 PRP(ml) 50 50 50 50 50 50 50 50 50 50 Bone 10 20 10 10 10 20 10 particles(ml) cranio- facial Bone 10 20 40 10 10 20 10 10 20 30 particles (ml)iliac Innovin (mg) 20 20 10 20 10 10 20 30 10 10 PepGen P-15 35 40 50 5050 60 65 70 75 75 (ml) Water (ml) 2 2 4 2 2 2 2 2 4 4

The bone generating product of said examples 1 to 19 having the form ofa gel can easily be implanted in a patient, for example in a humanpatient suffering a major maxillofacial atrophy. The bone generatingproduct of the invention can easily be compacted in recesses of bones,and can be easily be shaped.

The bone generating product of the invention was used for volunteerssuffering a major maxillofacial atrophy. Sinus lift grafts and on laygraft on the maxillofacial bone have been carried out. These tests haveshow a bone growth or the generation of bone where the bone generatingproduct of the invention was applied.

Example 20

A human bone was denatured and γ-irradiated so as to be prion free. Thebone was ground in particles having an average (by weight) of 0.2 mm.After drying, log of bone particles were dry mixed with 10 mg dryInnovin, so as to obtain a mixture of recombinant compound forgenerating thrombin, phospholipid and a high level of calcium containingcompound.

The so prepared mixture was then used for the preparation of a bonegenerating product of the invention.

The method of example 1 was repeated, except that the mixture 10 mgInnovin+10 g denatured bone particles was used instead of 10 mg Innovinalone, and except that a larger amount of sterile water was used (5-10ml), amount water adjusted so as to prepare a paste.

Example 21

Example 1 was repeated, except that before adding the recombinantthromboplastin, 200 μg Vibramycin® per ml mixture of PRP was added.

Example 22 Example 1 was repeated, except that before adding therecombinant thromboplastin, 100 μg Minocycline (Minocin®) per ml mixtureof PRP was added. Example 23

Example 1 was repeated, except that before adding the recombinantthromboplastin, 50 μg Minocycline (Minocin®) per ml mixture of PRP wasadded.

Example 24

Example 1 was repeated, except that before adding the recombinantthromboplastin, 20 μg Minocycline (Minocin®) per ml mixture of PRP wasadded.

Example 25

Examples 1 to 24 have been repeated, except that PepGen P-15 Flow wasused instead of PepGen P-15.

Example 26

Examples 1 to 24 have been repeated except that Innovin was replaced byanother recombinant tissue factor mixed with phospholipids. For example,the recombinant tissue factor is combined with at least a phospholipidselected from the group consisting of phosphatidylserine, derivativesthereof, phosphatidylserine having at least one fatty acid side chain,phosphatidylcholine, derivatives thereof, phosphatidylcholine having atleast one fatty acid side chain, and mixtures thereof. Preferably, therecombinant compound for generating thrombin is combined with at least aphospholipid selected from the group consisting of phosphatidylserine,derivatives thereof, phosphatidylserine having at least one fatty acidside chain, phosphatidylcholine, derivatives thereof,phosphatidylcholine having at least one fatty acid side chain, andmixtures thereof, the fatty acid side chain being selected from thegroup consisting of fatty acid chains with at least one double bond andwith 6 to 24 carbon atoms, most preferably with 16 to 18 carbon atoms.

According to a most preferred embodiment, the recombinant compound forgenerating thrombin is combined with a mixture of at least twophospholipids, a first phospholipid being selected from the groupconsisting of phosphatidylserine, derivatives thereof,phosphatidylserine having at least one fatty acid side chain, andmixtures therof, the fatty acid side chain of the phosphatidylserinehaving at least one fatty acid side chain being selected from the groupconsisting of fatty acid chains with at least one double bond and with 6to 24 carbon atoms, preferably 16 to 18, while the second phospholipidis selected from the group consisting of phosphatidylcholine,derivatives thereof, phosphatidylcholine having at least one fatty acidside chain, and mixtures thereof, the fatty acid side chain of thephosphatidycholine having at least one fatty acid side chain beingselected from the group consisting of fatty acid chains with at leastone double bond and with 6 to 24 carbon atoms.

The weight ratio recombinant tissue factor/phospholipids is comprisedfor example between 1:500 and 1:50, such as in this example between1:300 and 1:200, i.e. about 1:250.

In said examples, Innovin was replaced at the rate of 1.5 mg recombinanttissue factor+phospholipids per 10 mg Innovin used in examples 1 to 24.

Sealants were also prepared by mixing PRP, recombinant tissue factor ofHenogen S A and phospholipids, with and without antibiotics.

1. A tissue-generating product comprising a plasma matrix, one or moregrowth factors, at least one phospholipid and a protein scaffold for thegeneration of said tissue wherein the protein scaffold is a matrix ofcollagen, reticuline and/or elastine fibers or a precursor thereof. 2.The tissue-generating product according to claim 1, wherein theprecursor is tropocollagen or tropoelastine.
 3. The tissue-generatingproduct according to claim 1, wherein the plasma matrix is a coagulatedmatrix of platelet poor plasma comprising a platelet concentration lowerthan 500,000, 100,000 or 50,000 platelets per microlitre of the matrixforming agents.
 4. The tissue-generating product according to claim 1,wherein the growth factor is selected from the group consisting of thehuman (recombinant) tissue factor (rhTF), the human (recombinant)platelet-derived growth factor (rhPDGF), the human (recombinant)transforming growth factor (rhTGF), the human (recombinant) insulin-likegrowth factor (rhIGF), the human (recombinant) epidermal growth factor(rhEG), and the human (recombinant) hepatocyte growth factor (rhHGF). 5.The tissue-generating product according to claim 1, which furthercomprises at least one buffer and at least one antibiotic.
 6. Thetissue-generating product according to claim 1, wherein the tissue isskin or an epithelial tissue of the stomach.
 7. A kit for thepreparation of a tissue-generating product according to claim 1, whichcontains a vial containing human growth factors, the protein scaffoldelement being selected from the group consisting of collagen, reticulineand/or elastine fibers and their precursors or two distinct vials, afirst containing one or more growth factors, while the second vialcontaining a protein scaffold element selected from the group consistingof collagen, reticuline, and/or elastine fibers or their precursors, andoptionally a last vial which may contain at least one buffered agent andat least one antibiotic.
 8. A method for the preparation of atissue-generating product according to claim 1, in which: asubstantially homogenous mixture is formed by mixing a plasma matrixwith an effective amount of a protein scaffold element selected from thegroup consisting of collagen, reticuline and/or elastine fibers andtheir precursors; a growth factor and at least one phopholipid are addedand mixed to the mixture of the protein scaffold element and the plasmamatrix, and said mixture is kept under conditions for ensuring thecoagulation of the plasma matrix and the formation of thetissue-generating product.
 9. The method according to claim 8, whereinthe coagulation of the matrix is carried out in the presence of oxygenand substantially without stirring.
 10. The method according to claim 8,wherein the coagulation is carried out at a temperature comprisedbetween 35° and 40° C.
 11. (canceled)
 12. A method for generating atissue in a mammal patient, including a human in need thereof, saidmethod comprising the step of applying at the place where the tissue hasto be generated the tissue-generating product according to claim
 1. 13.A method for treating tissue damage in a mammal patient, includinghuman, comprising administering to the patient the tissue-generatingproduct of claim
 1. 14. The tissue-generating product according to claim2, wherein the plasma matrix is a coagulated matrix of platelet poorplasma comprising a platelet concentration lower than 500,000, 100,000or 50,000 platelets per microlitre of the matrix forming agents.
 15. Thetissue-generating product according to claim 2, wherein the growthfactor is selected from the group consisting of the human (recombinant)tissue factor (rhTF), the human (recombinant) platelet-derived growthfactor (rhPDGF), the human (recombinant) transforming growth factor(rhTGF), the human (recombinant) insulin-like growth factor (rhIGF), thehuman (recombinant) epidermal growth factor (rhEG), and the human(recombinant) hepatocyte growth factor (rhHGF).
 16. Thetissue-generating product according to claim 2, which further comprisesat least one buffer and at least one antibiotic.
 17. Thetissue-generating product according to claim 2, wherein the tissue isskin or an epithelial tissue of the stomach.
 18. A method for thepreparation of a tissue-generating product according to claim 2, inwhich: a substantially homogenous mixture is formed by mixing a plasmamatrix with an effective amount of a protein scaffold element selectedfrom the group consisting of collagen, reticuline and/or elastine fibersand their precursors; a growth factor and at least one phopholipid areadded and mixed to the mixture of the protein scaffold element and theplasma matrix, and said mixture is kept under conditions for ensuringthe coagulation of the plasma matrix and the formation of thetissue-generating product.
 19. The method according to claim 18, whereinthe coagulation of the matrix is carried out in the presence of oxygenand substantially without stirring.
 20. The method of claim 8, whereinthe coagulation is carried out at a temperature of about 37° C.
 21. Themethod of claim 9, wherein the coagulation is carried out at atemperature of about 37° C.